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Translation of abstract (English)

Despite extensive biochemical analysis of the formation of COPI coated vesicles, which revealed amongst other things the minimal machinery of COPI-vesicles (the small nuanine nucleotide binding protein (GNBP) Arf1, the coat complex coatomer and proteins of the p24-family) the mechanism of COPI-vesicle biogenesis is yet not fully understood. The small GNBP Arf1 in its GDP-bound form binds to membranes via dimeric p24-proteins (Gommel et al., 2001), where the GEF-mediated nucleotide exchange to GTP occurs. Exchanging GDP to GTP leads to conformational changes in Arf1, resulting in the exposure of its amphiphilic N-terminal alpha-helix with its myristoyl-anchor being inserted into the membrane (Antonny et al., 1997; Franco et al., 1996). Activated membrane-bound Arf1 dimerises, whereas a mutant of Arf1 – Arf1Y35A – is not able to dimerise and fails in generating free COPI vesicles (Beck et al., 2008). However, it is still unknown, at which step dimerisation occurs and how the dimer-interface looks like. The aim of this work was to elucidate the dimer-interface. First, a mutant with an aminoacid mutation in the putative dimer interface – Arf1Y35F – was generated and biochemically analysed. As it behaves like Arf1wt one can speculate that the bulky hydrophobic residue rather than the hydroxyl-group of Y35 is important for dimerisation. Secondly, to get insights into the dimer-interface, an N-terminal truncated version of Arf1 – Arf1NDelta17 – was purified in its GTP-bound form, chemically dimerised and crystallised. Analysis of the crystal structure, which describes an antiparallel double-helix, revealed, that chemically crosslinked Arf1 does not reflect the active Arf1-dimer found on membranes. As the N-terminus might play a role in dimerisation a third approach aimed at crystallising full length Arf1wt in its GTP-bound form in the presence of micelles, which did not succeed so far. The nucleotide exchange of GDP to GTP in Arf1 in the presence of micelles instead of membranes could be established.